Expression Profile Analysis Of Long Non-coding RNA In Gastrointestinal Stromal Tumor And Function And Mechanism Of LncRNA-DNM3OS In Regulating Malignant Progression Of GIST

Objective:The biological behavior of gastrointestinal stromal tumor(GIST)is variable,which can be benign or malignant.Previous studies have reported that long noncoding RNA(lnc RNA)can promote tumor occurrence,development and metastasis.However,its effect on the malignant progression of GIST and its underlying biological mechanism are not well understood.This study aims to screen out the lnc RNA involved in the malignant progression of GIST,in the hope of providing a new prognostic biomarker and therapeutic target for GIST.Materials and Methods:In this study,ten pairs of different risk GISTs and adjacent non-tumor tissues were collected for whole transcriptome sequencing to determine differentially expressed lnc RNA and m RNA.Then we conducted bioinformatics analysis,such as weighted correlation network analysis(WGCNA),Gene Oncology analysis(GO)and KEGG analysis,with the aim of exploring the function of biological processes,molecular functions and disease pathways involved of differentially expressed lnc RNA and m RNA.Candidate hub lnc RNA were selected by Oncomine analysis.Subsequently,the expression of a selected hub lnc RNA,DNM3 OS,was analyzed using Fluorescence in situ hybridization(FISH)staining in 251 GIST tissue samples,and its correlation with patients’ clinicopathological features and prognosis were also analyzed.The subcellular localization of DNM3 OS was detected by nucleocytoplasmic separation assay,quantitative PCR and FISH staining.DNM3 OS stableknockdown cell lines were constructed by transfecting lentivirus packged DNM3 OS sh RNA to GIST cells.The effects of DNM3 OS on the biological activities of GIST cells were examibed by CCK8 assays and immunofluorescence assays.The protein that directly bound to DNM3 OS were detected by RNA pull down assay combined with mass spectrometry,and verified by Western blot.The downstream target genes regulated by DNM3 OS were investiged by RNA sequencing and Western blot,and the regulatory network between DNM3 OS and downstream targets was constructed to explore the molecular mechanism of DNM3 OS regulating the malignant progression of GIST.Results:The GIST tissue samples were divided into three groups based on the modified NIH risk classification: low-risk GIST(LR,tumor size 2-5 cm,mitotic rate ≤ 5mitoses/50 HPFs),high-risk GISTs based on tumor size(HBS,tumor size > 10 cm and mitotic rate ≤ 5 mitoses/50 HPFs),and high-risk GISTs based on mitotic rate(HBM,tumor size 2–5 cm and mitotic rate > 10 mitoses/50 HPFs),and LR、HBS and HBM GISTs were collectd for RNA sequencing.Our results revealed a series of differentially expressed lnc RNA and m RNAs involved in the malignant transformation of GISTs.Venn diagram analysis identified 37,193,and 299 specifically upregulated lnc RNA in LR,HBS,and HBM GIST,respectively.The intersection between the lnc RNA upregulated in HBS and HBM GISTs revealed 118 lnc RNA co-upregulated in both HBS and HBM GISTs,which were associated with GIST malignant transformation.We focused on the lnc RNA involved in the most relevant signaling pathway identified by KEGG analysis of HBS/HBM coupregulated lnc RNA,the Hippo signaling pathway.The analysis identified 45 upregulated lnc RNA involved in the Hippo signaling pathway,and 12 clinically relevant lnc RNA were selected for further Oncomine analysis.Oncomine analysis revealed a tight association between clinical signatures in gastric cancers and DNM3 OS and suggested that DNM3 OS is a hub lnc RNA that is involved in the Hippo signaling pathway.Hence,we selected DNM3 OS for further investigation.The expression of DNM3 OS for 251 GISTs and corresponding adjacent normal tissues was tested by FISH staining,and high expression in 108 cases,low expression in 143 cases.The correlation analysis between DNM3 OS expression and various clinicopathological parameters indicated that DNM3 OS expression was positively associated with tumor size,mitotic count,NIH risk classification,and mutational status.Kaplan–Meier analysis revealed that higher DNM3 OS expression was associated with shorter progression-free survival(PFS)and overall survival(OS).Ensembl and NCBI gene database show that DNM3 OS is a 568-long antisense lnc RNA consisting of 4 exons,and was located in q24.3 region of human chromosome1.CPAT bioinformatics software predicted that DNM3 OS did not have protein-coding ability.Nucleo-cytoplasmic separation assay and FISH staining showed that DNM3 OS was mainly located in the nucleus in GIST cells.The down-regulation of DNM3 OS can obviously inhibit the proliferation and mitotic index of GIST 882 cells and GIST-T1 cells(P < 0.05).RNA pull-down assay combined with mass spectrometry and Western blot assay were applied to identify the proteins directly bind with DNM3 OS.The results showed that PARP1 could directly bind to DNM3 OS and promote the malignant progression of GIST.Transcriptome sequencing,quantitative PCR and Western blot assay were used to identify the downstream target of DNM3 OS and the signaling pathways involved.The results showed that DNM3 OS regulate the expression of GLUT4 and CD36,and then participate in Fox O,PPAR and AMPK signaling pathways to promote the malignant progression of GIST.Conclusion:There are a series of differentially expressed lnc RNA and m RNAs in the malignant progression of GIST,which may play an important role in the malignant progression.The expression level of lnc RNA-DNM3 OS in high malignant GIST was significantly higher than that in low malignant ones.The expression of DNM3 OS was significantly related to tumor size and mototic index,and Kaplan–Meier analysis revealed that higher DNM3 OS expression was associated with shorter PFS and OS,which could be used as a prognostic indicator for GIST.The down-regulation of DNM3 OS can obviously inhibit the proliferation and mitotic index of GIST cells.Mechanism studies indicated that DNM3 OS can directly target and bind to PARP1 to regulate the expression of GLUT4 and CD36,and then participate in Fox O signaling pathway to promote the malignant progression of GIST.These results suggested that DNM3 OS might be acted as a potential diagnostic and prognostic marker,and a novel therapeutic target for GIST.